Effects of sugar concentration on the electroporation, size distribution and average size of charged giant unilamellar vesicles.

We investigated the effects of sugar concentration on the electroporation, size distribution and average size of giant unilamellar vesicles (GUVs). GUVs were prepared from 40 mol% of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) and 60 mol% of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids. Pulsed electric field was applied to the 40%DOPG/60%DOPC-GUVs and it induced lateral electric tension (σc) in the membranes of vesicles. The σc-induced probability of rupture (Ppore) and the rate constant of rupture (kp) of GUVs under the sugar concentration, c = 40, 100 and 300 mM, were determined. Both the Ppore and kp increased with the increase of σc, but higher tension was required to generate the same values of Ppore and kp with increasing c. We also investigated average sizes of GUVs from the size distribution of vesicles under various sugar concentrations. With the increase of c, the peak of the size distribution histograms shifted to the region of smaller vesicles. The average size decreased 1.6-fold when c increased from 10 to 300 mM. These investigations help to understand various biomedical, biophysical, and biochemical processes in vesicles and cells. Electroporation, size distribution and average size of charged GUVs were investigated under various sugar concentrations. The sugar concentration influences the electroporation of vesicles and the average size of GUVs.

Stretch-activated pore of the antimicrobial peptide, magainin 2.

Antimicrobial peptide magainin 2 forms pores in lipid membranes and induces membrane permeation of the cellular contents. Although this permeation is likely the main cause of its bactericidal activity, the mechanism of pore formation remains poorly understood. We therefore investigated in detail the interaction of magainin 2 with lipid membranes using single giant unilamellar vesicles (GUVs). The binding of magainin 2 to the lipid membrane of GUVs increased the fractional change in the area of the membrane, δ, which was proportional to the surface concentration of magainin 2, X. This indicates that the rate constant of the magainin 2-induced two-state transition from the intact state to the pore state greatly increased with an increase in δ. The tension of a lipid membrane following aspiration of a GUV also activated magainin 2-induced pore formation. To reveal the location of magainin 2, the interaction of carboxyfluorescein (CF)-labeled magainin 2 (CF-magainin 2) with single GUVs containing a water-soluble fluorescent probe, AF647, was investigated using confocal microscopy. In the absence of tension due to aspiration, after the interaction of magainin 2 the fluorescence intensity of the GUV rim due to CF-magainin 2 increased rapidly to a steady value, which remained constant for a long time, and at 4-32 s before the start of leakage of AF647 the rim intensity began to increase rapidly to another steady value. In contrast, in the presence of the tension, no increase in rim intensity just before the start of leakage was observed. These results indicate that magainin 2 cannot translocate from the outer to the inner monolayer until just before pore formation. Based on these results, we conclude that a magainin 2-induced pore is a stretch-activated pore and the stretch of the inner monolayer is a main driving force of the pore formation.

Rate constant of tension-induced pore formation in lipid membranes.

We investigated the effects of tension induced by micropipet aspiration on giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). We analyzed the time course of the fraction of intact GUVs among all of the GUVs under constant tension σ and obtained the rate constants of pore formation kp(σ). To determine kp, we developed an approach using the mean first passage time. The fitting of the theoretical curves of kp versus σ to the experimental data determined the line tension of a prepore, Γ. The value of Γ of a DOPG/DOPC bilayer was smaller than that of a DOPC bilayer.

Effects of cholesterol on the size distribution and bending modulus of lipid vesicles.

The influence of cholesterol fraction in the membranes of giant unilamellar vesicles (GUVs) on their size distributions and bending moduli has been investigated. The membranes of GUVs were synthesized by a mixture of two elements: electrically neutral lipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol and also a mixture of three elements: electrically charged lipid 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), DOPC and cholesterol. The size distributions of GUVs have been presented by a set of histograms. The classical lognormal distribution is well fitted to the histograms, from where the average size of vesicle is obtained. The increase of cholesterol content in the membranes of GUVs increases the average size of vesicles in the population. Using the framework of Helmholtz free energy of the system, the theory developed by us is extended to explain the experimental results. The theory determines the influence of cholesterol on the bending modulus of membranes from the fitting of the proper histograms. The increase of cholesterol in GUVs increases both the average size of vesicles in population and the bending modulus of membranes.

Electrostatic interaction effects on the size distribution of self-assembled giant unilamellar vesicles.

The influence of electrostatic conditions (salt concentration of the solution and vesicle surface charge density) on the size distribution of self-assembled giant unilamellar vesicles (GUVs) is considered. The membranes of GUVs are synthesized by a mixture of dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine in a physiological buffer using the natural swelling method. The experimental results are presented in the form of a set of histograms. The log-normal distribution is used for statistical treatment of results. It is obtained that the decrease of salt concentration and the increase of vesicle surface charge density of the membranes increase the average size of the GUV population. To explain the experimental results, a theory using the Helmholtz free energy of the system describing the GUV vesiculation is developed. The size distribution histograms and average size of GUVs under various conditions are fitted with the proposed theory. It is shown that the variation of the bending modulus due to changing of electrostatic parameters of the system is the main factor causing a change in the average size of GUVs.

Deformation and poration of giant unilamellar vesicles induced by anionic nanoparticles.

The interaction of anionic magnetite nanoparticles (MNPs) of size 18 nm with negatively charged giant unilamellar vesicles (GUVs) formed from a mixture of neutral dioleoylphosphatidylcholine (DOPC) and negatively charged dioleoylphosphatidylglycerol (DOPG) lipids has been investigated. It has been obtained that NPs induces the deformation of spherical GUVs. The reaction of other GUVs on NPs consists in the appearance of pores in their membranes. We focused the effect of electrostatics on the interaction of charged membranes with MNPs. To study the influence of the surface charge of GUVs on the processes under consideration, we varied the fraction of DOPG in the vesicles from 0 to 100%. We examined the influence of salt concentration in the range of 50-300 mM NaCl concentration. To describe the degree of deformation, a special parameter compactness was introduced. The pore formation in the membranes of GUVs was investigated by the leakage of sucrose. The compactness increases with time and also NPs concentration. The fraction of deformed GUVs increases with the increase of surface charge density of membranes as well as the decrease of salt concentration in buffer. The value of compactness for neutral membrane is 1.25 times higher than that of charged ones. The fraction of deformed GUVs become constant after 20 min, however it increases with NPs concentration. The time taken for stochastic pore formation is less for charged membrane than neutral one. The physical mechanism explaining the experimental results obtained in these investigations.

Kinetic pathway of antimicrobial peptide magainin 2-induced pore formation in lipid membranes.

The pore formation in lipid membranes induced by the antimicrobial peptide magainin 2 is considered to be the main cause for its bactericidal activity. To reveal the mechanism of the pore formation, it is important to elucidate the kinetic pathway of magainin 2-induced pore formation in lipid membranes. In this report, to examine the change in pore size over time during pore formation which can monitor its kinetic pathway, we investigated the rate of the leakage of various sized fluorescent probes through the magainin 2-induced pores in single giant unilamellar vesicles (GUVs) of 50% dioleoylphosphatidylglycerol (DOPG)/50% dioleoylphosphatidylcholine (DOPC) membrane. Magainin 2- induced leakage of Texas-Red dextran 10,000, Texas-Red dextran 3000, and Alexa-Fluor trypsin inhibitor occurred in two stages; a transient rapid leakage in the initial stage followed by a stage of slow leakage. In contrast, magainin 2 induced a transient, but very small (10-20%), leakage of fluorescent probes of a larger size such as Texas-Red dextran 40,000 and FITC-BSA. These results indicate that magainin 2 molecules initially induce a large, transient pore in lipid membranes following which the radius of the pore decreases to a stable smaller size. We estimated the radius of these pores, which increases with an increase in magainin 2 concentration. On the basis of these data, we propose a hypothesis on the mechanism of magainin 2-induced pore formation.

Cationic DMPC/DMTAP lipid bilayers: local lateral polarization of phosphatidylcholine headgroups.

We investigated the effect of dimyristoyltrimethylammonium propane (DMTAP) charge on area per molecule of mixed DMTAP/dimyristoylphosphatidylcholine (DMPC) bilayers in a simple model. Assuming that trimethylammonium (TAP) charge causes lateral polarization of neighboring PC molecules, we analyzed variation in area per molecule as the mole fraction of TAP increases. The theoretical predictions obtained in the present study are consistent with results of a recent molecular dynamics simulation study (Gurtovenko et al. Biophys. J. 2004, 86, 3461).

Electrostatics explains the shift in VDAC gating with salt activity gradient.

We have analyzed voltage-dependent anion-selective channel (VDAC) gating on the assumption that the states occupied by the channel are determined mainly by their electrostatic energy. The voltage dependence of VDAC gating both in the presence and in the absence of a salt activity gradient was explained just by invoking electrostatic interactions. A model describing this energy in the main VDAC states has been developed. On the basis of the model, we have considered how external factors cause the redistribution of the channels among their conformational states. We propose that there is a difference in the electrostatic interaction between the voltage sensor and fixed charge within the channel when the former is located in the cis side of membrane as opposed to the trans. This could be the main cause of the shift in the probability curve. The theory describes satisfactorily the experimental data (Zizi et al., Biophys. J. 1998. 75:704-713) and explains some peculiarities of VDAC gating. The asymmetry of the probability curve was related to the apparent location of the VDAC voltage sensor in the open state. By analyzing published experimental data, we concluded that this apparent location is influenced by the diffusion potential. Also discussed is the possibility that VDAC gating at high voltage may be better described by assuming that the mobile charge consists of two parts that have to overcome different energetic barriers in the channel-closing process.